High density lipoproteins (HDL) are promising targets for pharmacological therapy of cardiovascular disease (CVD). Whether HDL itself directly prevents CVD or acts as a platform for attachment of protective antiinflammatory or antioxidant proteins, knowledge of HDL structure is important. The goal of the current proposal is to use a synergistic combination of direct experimental methods and computer simulations to understand the role of apolipoprotein A-I (apoA-I) dynamics in two important biological functions of HDL: i) activation of the enzyme lecithin:cholesterol acyl transferase (LCAT), the enzyme responsible for converting nascent (discoidal) HDL to circulating (spheroidal) HDL during HDL assembly, an important step in reverse cholesterol transport (RCT) and ii) HDL remodeling, also important in HDL assembly and RCT. Since apoA- I/HDL is a soft form of condensed matter easily deformable by thermal fluctuations, a more complete understanding of HDL will require innovative approaches. In principle, our proposed use of a synergistic combination of experimental methods and computer simulations can contribute significantly to understanding HDL structure and dynamics. Based upon our recent molecular dynamics (MD) simulations of HDL, we propose three working hypotheses: 1) A stochastic cloud of intrahelical and interhelical salt bridges, respectively, provide a spring-like elasticity (molecular "Slinky") and stickiness (molecular "Velcro") to apoA-I on HDL particles. 2) The terminal domains of apoA-I on HDL represent a remodeling-switch that regulates exchange of polar lipids and creates a hot spot with high affinity for other apolipoproteins and antiinflammatory and antioxidant proteins. 3) The pairwise antiparallel helix 5 domain of apoA-I creates an amphipathic presentation tunnel for migration of hydrophobic acyl chains and polar hydroxyl groups of unesterified cholesterol from nascent HDL to the active site of LCAT. To test these hypotheses we propose two specific aims: 1) To determine the role of the terminal overlap domain of apoA-I in nascent HDL remodeling. To achieve this aim, we will: i) Use our MD results to design experimental tests by site-directed mutagenesis of molecular models for fusion, exchange, membrane interactions and protein-binding affinity, focusing on the N- terminal "sticky" putative fusion domain and the C-terminal "promiscuous" helix 10 putative exchange domain. ii) Use all atom and coarse grained models of apoA-I/HDL ensembles (native and mutated) to further test the polar lipid remodeling-switch hypothesis by MD simulations. 2) To test the role of the central domain of apoA-I in LCAT activation. The acyl chain and UC presentation tunnel hypothesis will be tested experimentally by site-directed mutants designed on the basis of all atom and coarse grained MD simulations. Because of detailed predictions of lipid-associated apoA-I structure, the combination of wet lab approaches with molecular simulations that we propose and for which we are uniquely positioned can provide a molecular roadmap for future research into molecular mechanisms of HDL structure-function and dynamics. PUBLIC HEALTH RELEVANCE: HDL, the good cholesterol, is an important target for future drugs to prevent heart attacks. Unfortunately, all recent attempts at new HDL-targeted drug development have been unsuccessful. The combination of computer and molecular biology studies of HDL that we propose, a combination unique to our laboratory, provides a molecular blueprint for future drug development aimed at HDL.